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Primerjava testov za testiranje celičnih linij na prisotnost mikoplazem
ID Poljanšek, Tadeja (Author), ID Oven, Irena (Mentor) More about this mentor... This link opens in a new window

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Abstract
Mikoplazme so najmanjši in najpreprostejši prokarionti, ki pogosto okužijo celične kulture v laboratoriju in vplivajo na fiziologijo in metabolizem evkariontskih celic. Z različnimi testi, ki so vključevali PCR, barvanje DNA in komercialne teste, smo preverili prisotnost mikoplazem v celičnih kulturah v našem laboratoriju. Izkazalo se je, da je kar nekaj najpogosteje uporabljenih celičnih linij okuženih z mikoplazmami. Ker nas je zanimalo, s katero vrsto mikoplazem so okužene, smo pomnoženo zaporedje ribosomalne DNA mikoplazem sekvencirali in primerjali s sekvencami že znanih mikoplazem. Ugotovili smo, da so preverjene celične kulture okužene z Mycoplasma hyorhinis. Mikoplazme smo iz okuženih kultur poskusili odstraniti z dodatkom antibiotika gentamicina, ki smo ga dodali v visoki koncentraciji direktno v celično gojišče, vendar so celice ostale okužene, saj je M. hyorhinis nanj verjetno odporna. Za najbolj uporabno metodo detekcije mikoplazem v celičnih kulturah se je izkazala verižna reakcija s polimerazo, saj je bila najbolj zanesljiva in cenovno primerna, hkrati pa preprosta in primerna za analizo več vzorcev naenkrat.

Language:Slovenian
Keywords:mikoplazme, Mollicutes, celične linije, celične kulture, okužba, testiranje okuženosti, Mycoplasma hyorhinis, PCR, Hoechst 33342, sekvenciranje, zdravljenje, okužene linije
Work type:Master's thesis/paper
Typology:2.09 - Master's Thesis
Organization:BF - Biotechnical Faculty
Publisher:[T. Poljanšek]
Year:2020
PID:20.500.12556/RUL-114137 This link opens in a new window
UDC:602.3:579.887:576.32/.36:579.24:57.086(043.2)
COBISS.SI-ID:18059779 This link opens in a new window
Publication date in RUL:19.02.2020
Views:927
Downloads:181
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Secondary language

Language:English
Title:Comparison of tests for mycoplasma detection in cell cultures
Abstract:
Mycoplasmas are the smallest and simplest prokaryotes, which often infect cell cultures in laboratories and have an effect on their physiology and metabolism. We used different tests, including PCR, DNA staining and commercial kits, to inspect our cell cultures for mycoplasma contamination. We have shown, that some of our most frequently used cell cultures are contaminated with mycoplasmas. To find out the species of contaminating mycoplasmas, we used sequencing and compared our sequence to known species. All checked cell cultures were contaminated with Mycoplasma hyorhinis. To eradicate the contamination, we added antibiotic gentamycin in high concentration directly to the cell medium, but the cells remained mycoplasma contaminated, probably due to antibiotic resistance. The Polymerase chain reaction turned out to be the most useful method for detection of mycoplasmas in cell culture because of its reliability, price, simplicity and possibility of analysing more samples at the same time.

Keywords:mycoplasma, Mollicutes, cell lines, cell cultures, mycoplasma contamination, contamination detection, Mycoplasma hyorhinis, PCR, Hoechst 33342, sequencing, elimination treatments

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